Tuberculosis is a virulent human disease caused by the bacterium Mycobacterium tuberculosis that usually presents as a lung infection. It is highly infectious and if left untreated can be fatal. While the development of antibiotics has significanty decreased morbidity and mortality, M. tuberculosis is capable of establishing latent infections that are resistant to treatment and may eventually reactivate. In addition, M. tuberculosis can spread to and infect every tissue type, resulting in extrapulmonary infections that are extremely difficult to diagnose and treat. Understanding the pathogenesis of this important human disease and the mechanism by which the bacteria is able to spread within a patient and establish secondary sites of infection is critical to improving therapeutic options against extrapulmonary infections and potentially preventing latency and reactivation. We propose to investigate M. tuberculosis dissemination and extrapulmonary spread using a genetic approach. We will first test selected candidate mutants in the PknD signaling pathway, which we have demonstrated is important to M. tuberculosis dissemination, and identify additional genetic factors involved in dissemination by performing a Tn-seq screen to identify mutants that are unable to disseminate to the spleen. We will then characterize these mutants using a technique known as reporter enzyme fluorescence (REF). REF imaging relies on the activation of fluorescent probes by a beta-lactamase that is endogenously expressed by all strains of M. tuberculosis, allowing us to image dissemination mutants without performing any further genetic manipulations. This technology will allow us to determine the sites of infection and bacterial burden in each animal individually without sacrificing the animals, allowing us to image the same animal at multiple timepoints and follow the progress of an infection in vivo. We will analyze each mutant identified in our screen using REF imaging to confirm the dissemination defect and characterize the temporal kinetics and progression of M. tuberculosis extrapulmonary spread over the course of an infection. Finally, we will use an in vitro epithelial/endothelial cell bilayer model to investiate the cellular mechanisms by which M. tuberculosis disseminates across the pulmonary epithelia into the blood stream and lymphatic system and analyze the role of each dissemination gene in this process. We expect to gain insight into the molecular mechanisms of M. tuberculosis extrapulmonary spread by identifying the bacterial factors involved and determining their function and role in this critical process. This will increase our understanding of M. tuberculosis pathogenesis and will facilitate the design of targeted therapeutic interventions against tuberculosis.